Project summary The hippocampus is important for learning and memory and is highly susceptible to aggregation of microtubule- associated protein tau (MAPT) and neurodegeneration. Hippocampal and neocortical atrophy in Alzheimer?s disease (AD) brains demonstrates degeneration predominantly in large glutamatergic pyramidal neurons in association cortices while inhibitory interneurons and primary cortices are resistant to MAPT accumulation and degeneration. However, the molecular mechanisms that cause damage and death of susceptible neurons are not understood. Developing a better understanding of the molecular mechanisms causing vulnerability of excitatory neurons to damage and identifying pathways that regulate tau-mediated neurodegeneration will be essential to unraveling the pathogenesis and progression of AD and identifying potential therapeutic targets. The primary goal of this proposal is to identify pathways that make excitatory neurons susceptible to tau accumulation and neurodegeneration, and identify potential therapeutic targets for AD. One important limitation is the cellular heterogeneity of the mammalian brain. To overcome the cellular heterogeneity, this proposal will innovatively use single cell RNA sequencing in fresh AD human brain tissue and viral translating ribosome affinity purification (vTRAP) in a mouse model of tauopathy to generate transcriptional profiles of excitatory and inhibitory neurons from vulnerable and resilient regions of the brain in the context of aging and neurodegeneration. Global gene co-expression networks for excitatory and inhibitory neurons will be constructed through Weighted Interaction Network Analysis (WINA) and Multiscale Embedded Gene co-Expression Network Analysis (MEGENA). WINA and MEGENA derived modules will then be associated with AD and the top key drivers of the modules most associated with AD will become the candidate targets for experimental validation. We will also identify distinct and intersecting pathways from glutamate and tau mediated toxicities specifically in the pyramidal neurons of CA1 and CA3 regions of the hippocampus. Bacterial artificial chromosome TRAP (BAC-TRAP) reporter mouse lines in conjunction with models of glutamate dyshomeostasis (EAAT2-/-) and mutant human tau (P301S) will be used to generate translational profiles of CA1 and CA3 regions of the hippocampus at various stages of disease progression. Further, we will also evaluate the role of EAAT2, the major glutamate transporter, in tau accumulation, trans synaptic tau spread, immune dysfunction and neurodegeneration, and its potential as a therapeutic target using genetic (viral vector) and chemical (riluzole) approaches. This proposal will provide novel insights into the molecular mechanisms of excitatory neuronal susceptibility and resilience of inhibitory neurons in AD, identify potential new therapeutic targets for tau-mediated neurodegeneration and provide a mechanistic understanding of glutamate transporter EAAT2 as a mediator of inflammation and spread of tau pathology. A better understanding of the underlying molecular mechanisms of AD is crucial for the development of novel and more effective therapeutic targets. This proposal has the potential to make a significant impact to the field of AD by uncovering novel mechanisms of disease and identification of specific therapeutic targets.